Plasma and Platelet Tissue Factor Pathway Inhibitor (TFPI) in Blood Donors and in People with Hemophilia

Introduction: TFPI is an anticoagulant protein produced primarily by endothelium and megakaryocytes. It circulates in plasma as TFPIα (“free” TFPI; “full-length” TFPI) and as variably truncated forms bound to lipoproteins. TFPIα is also within platelets, and platelet TFPI promotes blood loss in a tail bleeding assay in Factor VIII- deficient mice (Maroney et al., PNAS 2012;109(10):3927-31). Furthermore, evidence suggests that inhibition of TFPI may represent an attractive treatment option for people with hemophilia (Hilden et al., Blood 2012;119(24):5871-8). Plasma and platelet TFPI in people with hemophilia were measured to determine the contribution of these TFPI pools to the severity of this bleeding diathesis, and to aid in the dosing of anti-TFPI compounds. Plasma and platelet TFPI were also measured in a large cohort of blood donors, to determine the demographic factors affecting these pools of TFPI.

Methods: High affinity monoclonal antibodies were used to develop ELISAs for plasma and platelet TFPI. Plasma total TFPI and TFPIα, and platelet TFPI, were determined in 427 blood donors (209 male, age 18-87; 218 female, age 19-89) and 58 people with severe hemophilia (47 hemophilia A, 11 hemophilia B). Data obtained from blood donors was stratified by gender, age, and oral contraceptive (OC) use, and reference ranges established (Table 1).

Results: Plasma total TFPI correlated with that obtained with a commercially available ELISA (r² = 0.75) while TFPIα did not (r² = 0.29). The current TFPIα assay uses a high affinity anti-K3 antibody with almost no dissociation, which may account for this discrepancy. The reference ranges for platelet and plasma TFPI are broad, varying approximately 5-fold. In males, platelet and plasma TFPI were not affected by age. In females, plasma TFPIα increases by ≈1% per year. Females have ≈10% lower plasma total TFPI than males, and ≈10% higher platelet TFPI. OC users have decreased plasma total TFPI and TFPIα compared to non-OC users. Interestingly, platelet TFPI in females did not vary by age or OC use. Preliminary analysis of data from people with hemophilia demonstrated that plasma total TFPI in either hemophilia cohort was not statistically different from male blood donors (Table 2). Plasma TFPIα in people with hemophilia A was approximately 17% lower than male blood donors (Table 2). A similar decrease was observed in people with hemophilia B when compared to male blood donors, though the difference did not reach statistical significance. Platelet TFPI in male blood donors and people with either form of hemophilia was not statistically different.

Conclusions: Robust immunoassays for the measurement of plasma and platelet TFPI were developed and validated. Poor correlation between the current TFPIα assay and other established assays suggests that the plasma TFPIα concentration may be underestimated in previous studies. The current study is the first to determine platelet TFPI concentration in a large cohort of normal donors, and in people with hemophilia. Plasma TFPI is affected by gender, age, and oral contraceptive use, whereas platelet TFPI is affected by gender only. People with hemophilia have normal plasma total TFPI and platelet TFPI, and mildly decreased plasma TFPIα. This data will be used to investigate the contribution of plasma and platelet TFPI to the bleeding diathesis in people with hemophilia, and to develop dosing strategies for the administration of anti-TFPI compounds that may be used in their treatment.